We thank Dr. Jordi Ochando from the Flow Cytometry Shared Resources at the Mount Sinai Medical Center for their assistance in flow cytometry analysis. We thank Dr. Rumana Huq from the Microscopy Shared Resource Facility at the Mount Sinai Medical Center for their imaging assistance. The authors thank the TJ Martell Foundation for its support in this project.

이 프로토콜은 기관 연구 윤리 보드에서 승인을 우리의 기관에서 수행과 인간의 복지에 대한 모든, 기관, 국가, 국제 지침을 준수하고 있습니다. 고급 전립선 암 환자에서 말초 혈액 1. 컬렉션 참고 : 전이성 전립선 암 환자를 선택합니다. 기록 된 환자의 동의를 얻어 격리 연령 이전 화학 요법과 호르몬 치료를 포함하여 환자의 임상 적 특징을 기록한다. 전이성 질환을 가진 환자는 잠재적으로 말초 혈액에서 CTCS의 가장 높은 농도를해야합니다. <ol><li> 동의 후 적절한 임상 시험 심사위원회가 프로토콜을 승인하에 혈액 샘플을 수집합니다. 라텍스 장갑 및 절차 전반에 걸쳐 실험실 코트를 착용 할 것. </li><li> 바늘 홀더에 25 G 나비 바늘과 튜브를 연결합니다. 의 영역을 청소하기 위해 알코올 면봉을 사용하여이두와 팔뚝 사이의 전주 정맥 후 환자의 상완에 지혈대를 적용합니다. </li><li> 전주 정맥에 나비 바늘을 삽입하고 수집을 시작하기 위해 바늘 홀더에 혈액 수집 튜브를 밀어 넣습니다. 혈액 응고를 방지하기 위해 에틸렌 디아민 테트라 아세트산 (EDTA)을 함유 수집 튜브에 혈액의 약 10 ㎖를 수집한다. </li><li> 환자에서 나비 바늘을 제거하고 1 분 동안 정맥 천자의 사이트를 통해 멸균 거즈 패드에 압력을 적용합니다. 멸균 붕대로 커버 사이트. </li></ol> 단핵 세포의 분리 (2) 주 : 1 단계에서 수집 된 혈액 단핵 CTCS 이외에 말초 혈액 단핵 세포 (PBMC) (예 : 림프구 및 단핵구)를 포함 할 것이다. <ol><li> : 1 비율 피펫 전체 5 ㎖의 혈청 학적 피펫 50ml의 폴리스티렌 원뿔 튜브에 혈액과는 1 행크스 균형 소금 솔루션 (HBSS)를 추가합니다. 조심스럽게 피펫 혼합물 균질화한다. </li><li> 빈 50 ml의 폴리스티렌 원뿔 관에 저속 밀도 구배 원심 분리하여 혈액 성분을 분리하기 위해 상업적으로 준비된 솔루션 (피콜 - 페이크)의 15 ML을 추가합니다. </li><li> 부드럽게 별개 상부층을 형성하는 단계에서 2.2 폴리스티렌 튜브에, 단계 2.1에서, 전혈 및 HBSS 혼합물을 피펫. 솔루션의 혼합을 최소화하기 위해 낮은 설정으로 설정 피펫이 효율적인 분리를 보장합니다. </li><li> RT에서 30 분 동안 400 XG (25 °의 C)에서 원심 분리한다. 가장 낮은 설정으로 감속 분리 후 솔루션의 혼합을 방지합니다. 가속도가 어떤 속도로 설정 될 수있다. </li><li> 원심 분리 후, 튜브의 하단에 상단과 분리 솔루션에 플라즈마 사이의 PBMC를하고 CTCS 끼워 층의 얇은 흰색 - 회색 스트라이프를 식별합니다. </li><li> 플라스틱 전송 피펫을 사용하여 50 ㎖ 폴리스티렌 튜브에 단계 2.5에서 흰색 - 회색 스트라이프에서 세포를 수집합니다. </li><li> PBMC를하고 CTCS와 C 50 ml의 폴리스티렌 원뿔 관에 HBSS 추가10 분 RT는 씻어 400 XG에 다시 entrifuge. </li><li> 씻어 실온에서 3 분 동안 400 XG에 다시 HBSS 원심 분리기 50 ml의 액체에 resuspend 펠렛을 가만히 따르다. </li><li> 상층 액을 버리고, 적혈구 용해 버퍼의 5 ㎖로 나머지 펠렛을 재현 탁하고 실온에서 5 분 솔루션을 품어. </li><li> RT에서 3 분 동안 400 XG에서 원심 분리하여 적혈구 용해 완충액을 제거한다. </li><li> 상층 액을 제거하고 종래 항체 염색 차단 10 % 소 태아 혈청 (FBS) 보충 된 1 ml의 PBS 1X에 펠렛을 재현 탁. </li></ol> 정렬 형광 활성 세포에 대한 CD45-FITC 항체 3. 염색 단핵 세포 (FACS) <ol><li> 혈구 또는 자동 세포 계수기를 사용하여 (단계 2.11)에서 실행 가능한 (트리 판 블루 음성) 세포의 수를 정량화. (10 % FBS와 PBS 1X)에 1 × 106 세포 / ㎖ 현탁액을 준비하고 1 시간 동안 얼음에 놓습니다. </li><li> 정량 배포두 튜브에 이전 단계에서 세포 현탁액. 제어 및 CD45 염색 등의 레이블 튜브. </li><li> 250 : 제어 튜브에서, (1)의 희석 IgG1κ-FITC (2.5 μg의 / ㎖)을 추가한다. CD45 염색 튜브에서, (1)의 희석 CD45 FITC (2.5 μg의 / ㎖) 차 항체 컨쥬 게이트를 추가한다 (250)와 30 분 동안 얼음에 세포 현탁액을 배양한다. CD45 항원 라벨링은 조혈 세포를 배제하기 위해 사용된다. </li><li> 4 ℃에서 3 분 동안 400 XG에서 세포를 원심 분리기, 상층 액을 버린다. </li><li> 멸균 1 X PBS로 각 펠렛을 일시 중단에 의해 두 번 세포를 씻으은 4 ℃에서 3 분 동안 400 XG에서 원심 분리 한 다음 10 % FBS와 보충. </li><li> 10 μg / ml의 농도 4 &#39;, 6-diamidino -2- 페닐 인돌 (DAPI)을 함유하는 PBS의 1 배의 1 ml의 용액 중에 세포를 재현 탁. </li><li> 12mm X 75mm의 폴리스티렌 튜브에 35 μm의 여과기 캡을 통해 최종 솔루션을 필터링합니다. </li></ol> 전립선 CTCS 4. 분리하여FACS 참고 : 라이브 CD45 부정적인 CTCS를 수집하는 사이토 흐름을 활용합니다. <ol><li> 레이블 튜브에서 세포를 사용하여 설정 보상 컨트롤 &quot;제어.&quot; </li><li> 파편과 적절한 앞​​으로 산란과 측면 산란 매개 변수를 사용하여 세포의 클러스터를 제외 게이트를 만듭니다. </li><li> 표지 된 튜브에서 세포 현탁액을 이용하여 FITC 게이트 확립 &quot;CD45-FITC를.&quot; </li><li> 태평양 블루 채널을 사용하여 게이트 가능한 (DAPI 음성) 세포. </li><li> 10 % FBS가 보충 된 로스웰 파크 메모리얼 연구소 (RPMI) 1640 2 ㎖를 함유하는 멸균 15 ml의 폴리스티렌 튜브에 원추형 CD45 네가티브 인구를 수집한다. </li><li> 10 % FBS가 보충 된 RPMI 500 μL - 3 분 동안 원심 분리기 (400)에서 정렬 XG 전립선 종양 세포 현탁액을, 다음 200에서 펠렛을 재현 탁. </li></ol> 이종 이식 성장의 마우스 및 모니터링에 CTCS 5. 주입 노트 :기관 동물 관리위원회에 의해 승인 된 프로토콜을 준수하여 모든 동물 절차를 실시한다. 이 프로토콜은 모든 관련 규제 및 제도 기관, 규정 및 지침에 따라 준수에 우리의 동물 관리위원회의 승인을 특정 동물을 사용 의정서에 따라 우리 기관에서 진행되고있다. <ol><li> 얼음에 1 비율 및 장소 : 1에서 세포 외 기질로 분류 전립선 종양 세포 현탁액을 섞는다. </li><li> 산소 / 분 1 L의 5 % (v / v)로 흡입 이소 플루 란을 공급 챔버 내에 피하 주입하기 전에 마우스를 마취. 마우스 각막과 발가락 반사의 손실을 확인하여 적절한 마취를 확인합니다. </li><li> 25 G 바늘과 1 ML의 주사기를 사용하여, 8~10주 세 남자 면역 결핍 마우스의 양쪽 상단 측면에 전립선 종양 세포와 세포 외 기질 세포 현탁액의 피하 250 μl를 주입. 주입 부피는 I 인 250 μL없이 CTC 농도 주입mportant 값. 마우스에 최대 SQ 정부는 더 이상 1 ml를하지 않습니다. &#160;</li><li> 피하 결절 밀도의 성장을위한 마우스 주사 부위의 주간 촉진을 수행함으로써 일주일에 두 번 마우스의 무게를 측정하여 마우스를 모니터링합니다. </li><li> 종양의 크기가 분자 분석 및 직렬 통로 충분한 종양 조직을 위해 0.5 cm 및 최대 직경 미만 1cm 때 경추 탈구 수확 피하 인간 전립선 암을 이종 이식 한 다음 이산화탄소 질식에 의해 쥐 안락사. 더 종양에​​도 불구하고 고통 또는 15 %의 체중 감소의 징후와 쥐를 안락사는 만져서입니다. </li></ol>

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The authors declare that they have no competing financial interests.

이 원고 CTCS에서 전립선 암 PDX 모델 생성 방법을 설명한다. PDX의 생성을위한 CTCS의 사용 기존의 방법에 비해 여러 가지 잠재적 모델 중요한 이점을 갖는다. 첫째, 말초 혈액에서 CTCS의 접근 컬렉션 다른 질병 단계에서 동일 환자에서 실험 모델의 생성을 가능하게한다. 둘째, 채혈은 종양 세포의 수집을위한 수술을 필요로 기존 방법에 비해 종양 세포를 분리하기 위해 안전하고 저렴한...

A correction was made to <a href="http://www.jove.com/video/53182">Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells</a>. There was a spelling error in one of the authors' surname. The author's name was corrected from:
Veronica Rodriquez-Bravo
to:
Veronica Rodriguez-Bravo

A correction was made to Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells.

Erratum: Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

환자 유래의 이종 이식은 암 연구에 사용되는 점점 더 인기를 실험 모델입니다. 이들은 생체 생물학적 경로, 전임상 약효 평가, 및 개인화 된 암 치료를위한 1,2- 아바타의 생성 특성에 사용될 수있다. 이전에, 다른 연구 그룹은 주입 또는 면역 저하 된 마우스 (1)에 단일 종양 세포 현탁액 또는 전체 종양 이식편을 주입함으로써 어느 PDX 모델을 개발 하였다. 이 PDX 모델은 신선한 고형 종양의 수술 수집, 악성 복수 또는 비용이 많이 드는 둘과 인성 사망률의 증가 위험에 환자를 노출하는 수술을받은 환자에서 흉막 삼출이 필요합니다. 암 연구 분야에서 최근의 발전은 상당한 종양 세포 순환의 검출, 분리 및 특성화되었다. 이들 종양 세포가 일차 종양 덩어리에서 탈출하고 순환을 입력그들은 전이 및 재발에 중요한 역할을하는 경우, 암의 가장 흔한 원인은 관련 사망률 3. 여러 고형암 유형에서 CTCS의 평가 및 특성 진단, 예후, 잔류 병 (3)를 모니터링하기위한 임상 정보를 제공하고 있습니다. 어느 물성, 바이오 마커의 발현 또는 CTCS의 기능적 특성에 의존하는 현재 사용되는 방법의 다양한 효율적 CTCS 4 분리하는데 사용될 수있다. 기존 거시적 CTC 분리 방법은 표면 분자에 대해 밀도 구배 원심 분리, 필터 기공 물리적 여과 및 분리를 포함한다. 가장 널리 사용되는 CTC 분리 방법론 CTCS 항체 기반의 캡처에 기초한다. 두 세포 표면 마커의 양성 및 음성 선택은 말초 혈액으로부터 CTCS를 분리하는데 이용 될 수있다. 말초 순환에 CTCS에 대한 긍정적 인 선택은 일반적으로 상피 마커 (예를 들어,는 EpCAM)를 사용하는CTCS하지만 조혈하지 세포에서 발현 다시. 이 방법의 단점은 전이 가능성이있는 CTCS 자주 상피 - 투 - 중간 엽 3 상피 표면 마커를 downregulates 전이 (EMT),받은 것입니다. 전이성 잠재력 CTCS를 분리하기 위해, 조혈 표면 마커 CD45을 채용 음성 선별 방법은, 백혈구의 정상 세포 집단 5 ​​사용될 수 고갈. 전립선 암은 가장 일반적으로 암 진단 및 남성 6의 암 관련 사망의 주된 원인이다. 종양 진행 및 공격성의 메커니즘은 완전히 이해되지 않으며, 따라서 생성 및 전립선 암의 분자 이질성 요점을 되풀이 실험 모델의 특성은 중요한 관심사이다. 전립선 암 PDX 모델왔다 immunocom에 인간 전립선 암 세포의 생착에 의해 이전에 생성 된약속 마우스 7,8. 그러나, 이러한 모델의 생성은 주로 병의 무통 성질에 기인 마우스에 면역이 전립선 암의 생착 낮은 속도에 의해 방해되어왔다. 최근 CTCS 유방암 9, 폐암 및 전립선 암 10 11 PDX 모델을 생성하는 데 사용되어왔다. 이러한 개념 증명 연구는 수술 시료 채취의 필요성 PDX 모델 독립적 발생의 가능성을 소개했다. 이 문서에서 우리는 구체적으로이 소설의 실험 모델의 생성하는 방법을 설명합니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640</td>
			<td>Gibco Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning Cell Gro</td>
			<td>21-031-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>35 &#181;m Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml polystyrene conical tube</td>
			<td>Crystalgen</td>
			<td>23-2263</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
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		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>d3571</td>
			<td></td>
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			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440</td>
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		</tr>
		<tr>
			<td>12 mm x 75 mm Polystyrene tubes with cell strainer cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Lavender Blood Collection Tubes with EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Winged Blood Collection Set with Push Button Retract Needle 23 gauge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer One Use Needle Holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Latex Tourniquet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Latex or non-latex gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alcohol swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x2 cotton gauze pads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive bandage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>25 gauge needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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</table>

generation of prostate cancer patient derived xenograft models from circulating tumor cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

이 프로토콜은 절연 CD45 네가티브 전립선 암에서 CTCS PDX 모델의 생성을 초래할 것이다. 우리의 프로토콜에서 사용되는 음성 선별 방법에 따라 그 DAPI 염색을 이용하여 사균을 배제 할 필요가있다. 유동 세포 계측법에 의해 검출 CD45 음성 세포의 비율은 가변적이며 환자 (도 1a)의 종양 부담에 따라 달라진다. 흰색 화살표 (도 1b)로 표시된 바와 같이 (세포 핵을 식별) CD45 및 DA...

Watch this Scientific Journal Video about Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells at JoVE.com

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

전립선 암 환자의 발생은 종양 세포를 순환에서 이종 이식 모델을 파생

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

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14.0K 조회수.  Icahn School of Medicine at Mount Sinai. This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

비디오: Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

기본 Preneoplastic 유방 상피 세포의 시간 경과 영상은 유방암의 유전자 변형 마우스 모델에서 파생

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

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Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

환자 유래 세포 배양 및 CD133의 절연<sup&gt; +</sup흑종에서&gt; Putative 암 줄기 세포

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

임상 및 전임상 연구 응용 프로그램에 대한 종양 세포 (CTC) 분석 실험을 순환 반자동의 적응

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

인간의 전립선 암 샘플에서 암 줄기 세포의 분리

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

환자와 인간의 내피 세포에서 파생 된 전립선 순환 종양 세포 사이에 E-셀렉틴 매개 상호 작용을 관찰하는 시험 관내 방법에

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

근육 영양 장애 환자에서 유도 만능 줄기 세포의 생성 : 소변 유래 세포의 효율적인 통합이없는 프로그램을 다시

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

질병 모델링을위한 근육 조직 검사에서 환자 유래 세포주의 분리 및 멸화

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

난소 암 종양 시작 세포의 생체 농축에

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

닭에서 인간의 다발성 골수종 이종 이식 모델의 설립은 종양의 성장, 침략과 혈관 신생을 공부하기

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.